Cytocapacity test for the prediction of the hematopoietic recovery, neutropenic fever, and antimicrobial treatment following high-dose cytotoxic chemotherapy

ABSTRACT

The present invention relates to a method for determining the hematopoietic cytocapacity of a subject comprising the steps of: (a) determining the amount of leukocytes present in a blood sample obtained from a subject, wherein said subject has been subjected to administration of a single dose of G-CSF and has been maintained for a time sufficient to allow mobilization or release of the leukocytes from hematopoietic production and storage tissues and sites of margination into the blood; and (b) determining the hematopoietic cytocapacity by assessing the amount of leukocytes determined in step (a) with the amount of leukocytes which have been mobilized or released in a control subject wherein said control subject is selected from the group consisting of subjects having (i) a high risk for a disease, disorder or complication associated with high-dose cytotoxic chemotherapy and/or hematopoietic cell transplantation, (ii) an intermediate risk for a disease, disorder or complication associated with high-dose cytotoxic chemotherapy and/or hematopoietic cell transplantation or (iii) a low risk for a disease, disorder or complication associated with high-dose cytotoxic chemotherapy and/or hematopoietic cell transplantation.

The present invention relates to a method for determining thehematopoietic cytocapacity of a subject comprising the steps of: (a)determining the amount of leukocytes present in a blood sample obtainedfrom a subject, wherein said subject has been subjected toadministration of a single dose of G-CSF and has been maintained for atime sufficient to allow mobilization or release of the leukocytes fromhematopoietic production and storage tissues and sites of marginationinto the blood; and (b) determining the hematopoietic cytocapacity byassessing the amount of leukocytes determined in step (a) with theamount of leukocytes which have been mobilized or released in a controlsubject wherein said control subject is selected from the groupconsisting of subjects having (i) a high risk for a disease, disorder orcomplication associated with high-dose cytotoxic chemotherapy and/or,hematopoietic cell transplantation, (ii) an intermediate risk for adisease, disorder or complication associated with high-dose cytotoxicchemotherapy and/or hematopoietic cell transplantation or (iii) a lowrisk for a disease, disorder or complication associated with high-dosecytotoxic chemotherapy and/or hematopoietic cell transplantation. Thepresent invention also relates to a method for selecting a suitableantimicrobial prophylaxis or therapy, a suitable prophylaxis or therapyfor neutropenic fever, a suitable amount of CD34⁺ cells fortransplantation, a suitable amount of a hematopoietic growth factor fora subject or a suitable level of supportive care. Finally, encompassedby the present invention is the use of leukocytes obtained from asubject for the preparation of a diagnostic composition for diagnosing asusceptibility for a disease, disorder or complication associated withhigh-dose cytotoxic therapy and/or hematopoletic cell transplantation insaid subject, wherein said subject has been subjected to administrationof a single dose of G-CSF and has been maintained for a time sufficientto allow mobilization or release of the leukocytes from hematopoieticproduction and storage tissues and sites of margination into the blood.

The myelosuppression and hematopoietic recovery after cytotoxicchemotherapy for malignancies is the major determinator of thetreatment-related morbidity and mortality (Bodey 1966; Link 1994). Thetransplantation of autologous hematopoietic stem and progenitor cellsharvested from bone marrow or the peripheral blood can rescue the hosthematopoietic functions after high-dose therapy. High-dose therapyshowed favorable disease control and survival in patients with lymphomaor multiple myeloma (Philip 1995; Attal 1996; Barlogie 1997; Lenhoff2000). The use of mobilized peripheral blood stem cells (PBSCs) hasalmost completely replaced the use of autologous bone marrow as a graftbecause of a faster engraftment (Beyer 1995; Schmitz 1996). The CD34⁺cell number in the PBSC graft is a marker for the stem and progenitorcell quantity and related with the hematopoietic recovery. Although thenumber of CD34⁺ cells can be precisely determined, a large variabilityin the prediction of the hematopoietic recovery remains (Bensinger 1995;Tricot 1995; Weaver 1995; Ketterer 1998). Host factors which play a rolein the hematopoietic recovery after cytotoxic therapy are poorly defined(Tricot 1995; Bolwell 1997).

Neutropenia after high-dose therapy usually is short with the use ofmobilized PBSCs, yet it is severe for some days. The majority ofpatients develop neutropenic fever indicating infection (Kolbe 1997;Reich 2001). The clinical course of infection is variable and in aproportion of the patients complications can become life-threatening.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growthfactor specific for neutrophil production and function. It increases theproliferation rate of neutrophil progenitor cells in the bone marrow andaccelerates the neutrophil maturation (Lord 1989). The majorapplications of G-CSF today are to reduce the risk of neutropenic feverand to mobilize PBSCs (Ozer 2000).

A risk assessment for a myelosuppressed subject, if possible, could beused to define different levels of supportive care, to stratify theapplication and dose of hematopoietic growth factors and intensity andduration of antimicrobial prophylaxis and therapy.

Thus, the technical problem underlying the present invention is toprovide means and methods for the prediction, diagnosis, prognosis andtreatment of diseases, disorders or complications associated withhigh-dose cytotoxic chemotherapy and/or hematopoietic celltransplantation whereby the aforementioned undesirable side effects areto be avoided, ameliorated or treated adequately.

The technical problem underlying the present invention is solved by theembodiments characterized in the claims.

Accordingly, the present invention relates to a method for determiningthe hematopoietic cytocapacity of a subject comprising the steps of:

-   -   (a) determining the amount of leukocytes present in a blood        sample obtained from a subject, wherein said subject has been        subjected to administration of a single dose of G-CSF and has        been maintained for a time sufficient to allow mobilization or        release of the leukocytes from hematopoietic production and        storage tissues or sites of margination into the blood; and    -   (b) determining the hematopoietic cytocapacity by assessing the        amount of leukocytes determined in step (a) with the amount of        leukocytes which have been mobilized or released in a control        subject wherein said control subject is selected from the group        consisting of subjects having (i) a high risk for a disease,        disorder or complication associated with high-dose cytotoxic        chemotherapy and/or hematopoietic cell transplantation, (ii) an        intermediate risk for a disease, disorder or complication        associated with high-dose cytotoxic chemotherapy and/or        hematopoietic cell transplantation or (iii) a low risk for a        disease, disorder or complication associated with high-dose        cytotoxic chemotherapy and/or hematopoietic cell transplantation        or a susceptibility therefor.

In one preferred embodiment, the invention relates to the above methodwherein in step (b), the control subject is a healthy subject. In afurther preferred embodiment of the invention, the assessment is carriedout by comparison of the leukocyte number determined in step (a) with astored number or range of leukocyte numbers of a control subject orcontrol subjects, which is (are) preferably (a) healthy subject(s).

Further, it is envisaged by the present invention that in step (b) theamount of leukocytes is present in a blood sample from the controlsubject in which said leukocytes have been mobilized or released.

The term “hematopoietic cytocapacity” refers to a value which ispredictive for diagnosis, prognosis and treatment of diseases, disordersor complications associated with high-dose cytotoxic chemotherapy and/orhematopoietic cell transplantation. Said value can be determined asdescribed below and in the accompanied Examples. It represents theleukocytes which are induced due to administration of a single dose ofG-CSF in a subject after cytotoxic therapies, such as high-dosechemotherapy. The hematopoietic cytocapacity expresses the impact of theinduced leukocyte peaks on a relative scale and/or categorization. Thehematopoietic cytocapacity may be calculated by categorizing thedistribution of the leukocyte peaks into <25. percentile, >25.percentile, <50. percentile, >50. percentile, <75. percentile and >75.percentile, or a transformation into relative values with respect to themedian of the distribution which can be set as 1.0; see alsoaccompanying examples.

The term “leukocytes” encompasses B and T lymphocytes, granulocytes,(eosinophiles, neutrophiles, basophiles), monocytes and macrophages aswell as precursors thereof or cells which are derived therefrom.

The term “subject” as used in accordance with this invention relates toanimals, preferably to vertebrates, and humans.

The term “administration” as used herein refers to all suitable modes ofadministration for a protein or polypeptide, such as G-CSF. Such modesof administration comprise intravenous and subcutaneous administrationof G-CSF. Moreover, the substance can be administered in combinationwith other substances either in a common pharmaceutical composition oras separated pharmaceutical compositions.

The term “a single dose” means that G-CSF is administered a defined timeinterval prior to the determination of the hematopoietic cytocapacity tothe patient and to further steps of medical treatment, such astransplantation of PBSCs or hematopoietic growth factor or antimicrobialtreatment to the subject. However, said term does not necessarilyrequire that G-CSF is only administered once, rather the single dose ofG-CSF can also be administered in several steps independently. Thesingle dose of G-CSF to be administered in accordance with the method ofthe invention may be given before, during, or after cytotoxic therapy.Preferably, the said single dose is administered after cytotoxic therapyhas been given. Administration of the said single dose of G-CSF duringcytotoxic therapy is preferably contemplated where the course of suchcytotoxic therapy comprises several administrations of the therapeuticagent(s) for such cytotoxic therapy, so that the said single dose ofG-CSF is administered when a first administration of a dose of atherapeutic agent for such cytotoxic therapy has been administered, butbefore the last dose of the same or another therapeutic agent for suchcytotoxic therapy has been administered. More preferably, said timeperiod is in the range of about 1 to about 120 hours. Still morepreferably, said time period is about 1 hour, about 2 hours, about 6hours, about 10 hours, about 12 hours, about 14 hours or about 18 hours.

In another embodiment of the invention, the single dose G-CSF may beadministered independently of a cytotoxic therapy and/or ofhematopoietic cell transplantation. The term “hematopoieticcytocapacity” may thus also refer to a value which is predictive fordiagnosis, prognosis and treatment of diseases, disorders orcomplications associated with infection. Said value can be determined asdescribed below and in the accompanied Examples, independently of priortreatment of the patient with cytotoxic therapy and/or hematopoieticcell transplantation. It represents the leukocytes which are induced dueto administration of a single dose of G-CSF in a subject. Thehematopoietic cytocapacity expresses the impact of the induced leukocytepeaks on a relative scale. The hematopoietic cytocapacity may becalculated by categorizing the distribution of the leukocyte peaks into<25. percentile, >25. percentile, <50. percentile, >50. percentile, <75.percentile and >75. percentile, or a transformation into relative valueswith respect to the median of the distribution which can be set as 1.0;see also accompanying examples. Further, the term “a single dose” maythus also mean that G-CSF is administered a defined time interval priorto the determination of the hematopoietic cytocapacity to the subjectindependently of further steps of medical treatment, such ashematopoietic growth factor or antimicrobial treatment or hematopoietictransplantation to the subject. Said term does not necessarily requirethat G-CSF is only administered once, rather the single dose of G-CSFcan also be administered in several steps. The single dose of G-CSF tobe administered in accordance with the method of the invention may begiven independently of cytotoxic therapy, and/or without any cytotoxictherapy.

The term “G-CSF” refers to polypetides or proteins having the biologicalactivity of granulocyte-colony stimulating factor as described inFilgrastim (r-metHu G-CSF) in clinical practice, Eds. George Morstyn,Second Edition 1998, Marcel Dekker Inc., New York; Kubota 1990; Asano1991; Welte 1996. The terms polypeptide and protein are usedsynonymously herein. Preferably, said G-CSF proteins or polypeptideshave an amino acid sequence of at least the mature sequence of G-CSF asshown in Genbank accession number CAA27291 (SEQ ID No: 1) or CAA27290(SEQ ID No: 2), wherein amino acids 1 to 30 correspond to the signalsequence and amino acids 31 to 207 correspond to the mature polypeptideresponsible for the biological activity of G-CSF. Moreover, the aminoacid sequence as shown in Genbank accession number CM01330 (SEQ ID No:3) or CAA01319 (SEQ ID No: 4) also correspond to the mature G-CSFpolypeptide. Therapeutically suitable G-CSF compositions arecommercially available and are described in detail below. The G-CSFpolypeptides or proteins, furthermore, encompass molecular variantshaving an amino acid sequence which is at least 70%, at least 80%, atleast 85%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to any one of theaforementioned sequences. In a preferred embodiment of the invention, aG-CSF variant having any of the sequences provided in EP0459630 may beused. Also, a modified sequence of G-CSF may be prepared in accordancewith the teachings of EP0459630, and the G-CSF so prepared may be usedin accordance with the present invention. Preferably, said molecularvariants having the biological activities of G-CSF as described above.Moreover, fragments of G-CSF having the biological activities of G-CSFas described above, are also encompassed in accordance with the methodof the present invention. Those fragments may comprise N- and C-terminaldeletions of the mature G-CSF polypeptides. Finally, also within thescope of the method of the present invention are fusion proteins of theaforementioned G-CSF proteins, molecular variants or fragments thereof.Said fusion proteins comprise in addition to the said G-CSF proteins,molecular variants or fragments thereof further amino acid sequenceswhich can be derived from proteins which are not related to G-CSF, suchas antibodies. However, said fusion proteins have to exhibit thebiological activities of G-CSF referred to herein. The present inventionencompasses also all suitable chemical modifications of theaforementioned proteins and polypeptides like pegylation. The process ofpegylation is known to the person of skill in the art. U.S. Pat. No.6,166,183 teaches a pegylated human G-CSF, its production and uses.

The proteins or polypeptides referred to above may be administered in asuitable diluent. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological saline, Ringers solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.Moreover, the said proteins or polypeptides may be co-administered withother therapeutics, such as standard medication, antimicrobials,monoclonal antibodies and other recombinant growth or developmentfactors.

The term “antimicrobial” is meant to include therapeutic agents for theprophylaxis or treatment of diseases, disorders and/or complicationsassociated with bacterial, fungal, viral, protozoal, or parasiticalinfections.

The term “a time sufficient to allow mobilization or release of theleukocytes” refers to the time which the leukocytes require to getactivated by G-CSF and the time which they require to be mobilized andto enter the blood. Said time window may vary between different classesof subjects and also between different individuals of a class ofsubjects. However, the person skilled in the art can easily adopt themethod of the invention without further ado.

The term “hematopoietic production and storage tissues and sites ofmargination” encompasses tissues and organs which are capable ofproducing, storing or harboring leukocytes. Said tissues or organs orsites comprise, inter alia, liver, bone marrow, spleen, lymphoid organs,lung, skin, vascular endothelium or the microcirculation.

The term “assessing” encompasses putting into relation the amount ofleukocytes determined in step (a) of the method of the invention withthe amount of leukocytes which have been mobilized or released in acontrol subject, wherein said control subject is known to have a definedrisk for a disease, disorder or complication associated with high-dosecytotoxic chemotherapy and/or hematopoietic cell transplantation or asusceptibility therefor. Preferably, said assessment is achieved bycomparing the said amounts of leukocytes or a statistic value derivedtherefrom. Suitable statistical values and the calculation thereof arewell known in the art and comprise, inter alia, the median anddistribution. Moreover, a detailed method how the assessment inaccordance with the present invention can be carried out is described inthe accompanied Examples. Most preferably, as shown in the accompaniedExamples, assessment is carried out by comparing the determined amountof leukocytes with categorized control groups having a defined risk forthe aforementioned diseases, disorders or complications orsusceptibilities therefor.

The term “control subject” refer to subjects for which the hematopoieticcytocapacity has been established already by the method of the inventionand for which the risk for a disease, disorder, complication orsusceptibility therefor has been known from previous sources of riskestimation based on experience. The term “control group” means a groupof control subjects which fit into the same risk category for a disease,disorder, complication or susceptibility therefor referred to inaccordance with this invention.

The term “high risk”, “intermediate risk” and “low risk” refers todifferences in the individual predisposition for developing a disease,disorder, complication or susceptibility therefor, preferably after asubject has been treated by one of the therapies referred to below, suchas high-dose chemotherapy. Said high, intermediate or low risk can bestatistically analyzed. Preferably, the differences between a subject ora group of subjects having a high, intermediate or low risk arestatistically significant. This can be evaluated by well known statistictechniques including Student's t-Test, Chi²-Test, Wilcoxon-Mann-WhitneyTest, Kurskal-Wallis Test or Fisher's exact Test, log-rank test,logistic regression analysis, or Cox models. Most preferably, the riskgroups are analyzed as described in the accompanied Examples wherebyexplorative data analysis is carried out and the risk groups are formedwith respect to the median, the 25% and the 75% percentiles. Based onthis analysis, relative scale cut-off values for risk group formationcan be defined. Within groups having received similar or identicalchemotherapies, also the absolute leukocyte values can be used ascut-offs. The term “similar or identical chemotherapies” preferablymeans such chemotherapies where essentially the same degree ofmyelosuppression is achieved at essentially the same time point.Differences in continuous variables of the groups are tested byWilcoxon-Mann-Whitney Test or Kurskal-Wallis Test depending on thenumber of groups to be compared. For nominal or ordered categories,Fisher's exact or Chi²-Test for trend are applied. Without further ado,the person skilled in the art can carry out multivariant analysis withstratified versions of the aforementioned tests or Cox models in orderto examine the independent impact of predictive factors and to establishthe different risk groups.

The term “disease, disorder or complication associated with high-dosecytotoxic chemotherapy and/or hematopoietic cell transplantation or asusceptibility therefor” comprises those diseases, disorders orcomplications which have been reported to be caused by or are associatedwith the procedure. Preferably, such diseases, disorders orcomplications comprise those explicitly mentioned below.

Advantageously, it has been found in accordance with the presentinvention that a predictive value for the risk assessment for diseases,disorders or complications associated with high-dose cytotoxicchemotherapy and/or hematopoietic cell transplantation, can be generateddue to determining the leukocyte peak which is generated by G-CSF in asubject which had been treated by cytotoxic chemotherapy. Within hoursafter G-CSF administration, leukocytes enter the circulation. Inaccordance with the present invention it has been observed that aleukocyte peak after a single dose of G-CSF given after high-dosetherapy and after further autologous transplantation of peripheral bloodstem cells (PBSCs) correlated with the hematopoietic recovery.Fluorescence in situ hybridisation (FISH) for XY chromosome on bloodleukocytes in sex-mismatched allogeneic hematopoietic transplantrecipients have demonstrated that the peripheral blood leukocytes atthis early time after transplantation and under G-CSF administration aremainly of host, and not transplant origin (Arseniev 1997). To furtherexclude any contribution of the autograft to this phenomenon, G-CSF hasbeen administered before autologous transplantation and it has beeninvestigated whether the induced transient leukocyte peaks were afunctional measure for the host hematopoietic capacity and would predictthe hematopoietic recovery and related variables after high-dosecytotoxic chemotherapy.

The value of the early leukocyte peaks in the circulation upon a singledose of G-CSF following to high-dose chemotherapy predicted the durationand severity of neutropenia and leukopenia and the recovery rate of hosthematopoiesis. The hematological parameters evaluated after thecytocapacity test are preferably one or more of the time to neutrophiland platelet recovery and also the duration of leukopenia <1000/μl, theduration of neutropenia <500/μl, and the residual neutrophil/leukocytelevel in the blood during myelosuppression. Surprisingly, thecorrelations between the cytocapacity test and these hematologicalparameters known to be associated with morbidity and mortality frommyelosuppression were highly significant. The cytocapacity testtherefore appeared to provide a measure of the capacity of hosthematopoiesis to overcome the effects of cytotoxic therapy. Preferably,at the time point of the cytocapacity test the blood cell counts stillare normal or close to normal and the test result can advantageously beconsidered for the management of the time period of myelosuppressionand/or the transplant.

The cytocapacity test may be used at a time point before, during, orafter cytotoxic therapy. The cytocapacity test yields a predictionregarding the assessment of the possibility of a disorder, complicationand/or disease. That prediction may advantageously be used in order todecide the stop of the therapy and/or modification of the treatment.Thus, after measuring very low cytocapacity test values, it may bedecided that no (further) cytotoxic therapy and/or transplantation isadministered. Alternatively, preferably when low to medium cytocapacitytest values are measured, it may be decided that the therapy should bemodified. For instance, the cytotoxic therapy may be modified, e.g. byreduction of the dose of one or more of the cytotoxic agents usedtherein, and/or by omission of at least one of such agents, and/or bychanging to another protocol of selection of cytotoxic agents and/ordose and administration regimen thereof, such that the cytotoxic therapyhas a less suppressive effect upon the patient's hematopoietic system.In addition, when a transplantation is administered, a higher number ofCD34+ cells may be contemplated to be necessary in order to reconstitutethe patient's hematopoietic system. Further alternatively or inaddition, the transplantation of allogeneic or autologous hematopoieticstem cells may be considered, in order to (faster) reconstitute thepatient's hematopoietic system. Still further, a standard antibiotictreatment usually given may be modified so as to include furtherantibiotics, different antibiotics, and/or higher doses of antibiotictreatment, in order to more effectively prevent and/or treat infectionand the associated complication, disorder, disease and/or condition. Theterm “antibiotic” is to be understood herein to comprise substanceseffective against bacteria, parasites, protozoa, fungi and/or viruses.

When comparing leukocyte numbers obtained from different patients orpatient groups, normalization of the values may be carried out. To thisend, a relative scale may be used, so that only the relative increase innumber is compared. Alternatively, or in combination, a categorizationmay be used, e.g., such as the categorization used in the Exampleshereinbelow wherein the values are categorized in quartiles. In anotherembodiment of the invention, the normalization is carried out on thebasis of the degree in myelosuppression achieved by cytotoxicchemotherapy and/or which is present in the patient and/or in thecontrol subject. Using this kind of normalization, different patientgroups with different degrees of myelosuppression at the time point ofassessing cytocapacity may advantageously be compared.

With increasing time interval between the start of cytotoxicchemotherapy and single dose G-CSF, the median of the test-inducedleukocyte peaks will be lower because of a decreasing availability ofmature blood cells. There is, however, no impact of this on the rate ofthe hematopoietic recovery. To take this into account, the leukocytepeaks can be evaluated on a relative scale separately for groups ofsimilar regimens. These relative values with respect to the median ofthe distribution constitute continuous cytocapacity test results asdiscussed, supra.

The cytocapacity test itself is independent from peripheral blood stemcell (PBSC) transplantation since the test is performed before thetransfusion of the cryopreserved PBSCs. The transplantation ofautologous PBSCs replenishes the chemotherapy-depleted progenitor cellpool. In multivariate analysis, the cytocapacity test was independentfrom the dose effect of PBSC CD34⁺ cells in prediction of thehematopoietic recovery.

The fast hematopoietic recovery with the use of mobilized PBSCs afterhigh-dose chemotherapy (Beyer 1995; Schmitz 1996) and the associatedgood tolerability and low treatment-related mortality below 5% hasspurred the interest in performing autografts on an outpatient basis(Meisenberg 1997; Herrmann 1999; Palumbo 1999). With a cytocapacitytest >1.0 and when at least a standard dose of CD34⁺ cells (>2.5×10⁶/kg)is transplanted, outpatient care as a possibility is suggested (see alsoExamples 3-5 hereinbelow). In this favorable group, there was an optimalneutrophil and platelet recovery, which was completed in 10 and 12 days,respectively. Therefore, in this group, the hematopoietic recoveryoccurred without delay and there was a significantly reduced risk ofinfection and a reduced requirement for antimicrobial therapy.Advantageously, with the availability of the cytocapacity test, thethreshold CD34⁺ cell number required to be transplanted to achieve afavorable hematopoietic recovery can be determined. Neutropenic feverfrequently is the first sign and hallmark of infection in cancerpatients after cytotoxic chemotherapy. It is associated with asignificant complication rate and usually leads to hospitalization andempiric broad-spectrum intravenous antimicrobial therapy as the standardof care (Pizzo 1993). Risk models incorporating a variety of clinicalcharacteristics have been developed to define the complication risk forpatients with neutropenic fever (Talcott 1992; Klastersky 2000). Lowrisk patients were candidates for empirical oral antimicrobial therapy(Kern 1999; Freifeld 1999) or outpatient treatment (Talcott 1994;Rubenstein 1993). Clearly, the intensity of chemotherapy is an importantrisk factor for neutropenic fever but also the individual susceptibilityto acquire an infection. Low lymphocyte counts after chemotherapy werepredictive for the occurrence of neutropenic fever (Blay, 1996). Theneutrophil nadir after a first cycle of chemotherapy predictedsubsequent neutropenia, chemotherapy dose reductions or treatment delaysoccurring in following cycles (Silber, 1998). The cytocapacity test,however, induces a rise in the blood leukocyte count prior tomyelosuppression. In the investigation underlying the present invention,the cytocapacity test was the major predictor for the development ofneutropenic fever, documented infection and the required intravenousantimicrobial therapy. Even when cases which had received ≦2.5×10⁶ CD34⁺cells/kg and which therefore constitute an unfavorable group wereexcluded from the analysis, the observed continuous direct relationbetween the cytocapacity test and the absence of neutropenic feverand/or documented infection and the inverse relation with therequirement for antimicrobial therapy did not change. The cytocapacitytest can provide the clinician with additional information regarding therisk of infection for an individual patient. The cytocapacity test alsoprovides an instrument for risk stratification.

Based on the investigation underlying the present invention, the methodof the present invention which can predict the hematopoietic recoveryindependently from the dose effect of transplanted CD34⁺ cells and canpredict the risk of infection following high-dose cytotoxic chemotherapyhas been established. Moreover, the cytocapacity test of the presentinvention can stratify the application and dose of hematopoietic growthfactors, the CD34⁺ cell number to be transplanted, the intensity andduration of antimicrobial prophylaxis and therapy and the level of carefor the myelosuppressed patient. Also encompassed by the invention is acytocapacity test in different forms of myelo- or immunosuppression dueto medical treatment, infection or primary and secondary bone marrowdiseases or disorders. Therefore, according to the present invention,there is contemplated a method of prophylaxis of an infection inmyelosuppressed subjects in general, by application of the cytocapacitytest and determination of a suitable antimicrobial, antifungal, and/orantiviral therapy in accordance with the results of the cytocapacitytest, optionally by considering further data about the general health ordisease state of the patient and optionally, his relatives. Theantifungal, antimicrobial, and/or antiviral therapy may be administeredas preventive and/or therapeutical treatment. For instance, in the caseof chronic infection, it may be decided that the patient is, based uponresults of the cytocapacity test and optionally, further data about thegeneral health or disease state of the patient and optionally, hisrelatives not able to effectively fight the infection. In consequence, atherapeutic treatment may be initiated.

Further, based upon a low results from the cytocapacity test, it may bedecided to treat the hematopoietic and/or immune system of the patient,rather than treat an infection or prevent an infection by prophylaxis.To this end, the transplantation of hematopoietic stem cells, or theadministration of hematopoietic growth factors, as single dose or asconsecutive doses or as a long-term treatment, may be considered. Onesuch hematopoietic growth factor is G-CSF. Other factors includeThrombopoietin, GM-CSF, Stem cell factor, Flt3 ligand, erythropoietin,KGF.

The present invention also relates to a method for selecting a suitableantimicrobial prophylaxis or therapy for a subject, wherein said methodcomprises the steps of the aforementioned method and the further step(c) selecting a suitable antimicrobial prophylaxis or therapy for saidsubject based on the results obtained in step (b).

The definitions and explanations of the terms made above apply mutatismutandis.

The term “suitable antimicrobial prophylaxis or therapy” encompassesmedical treatments which either prevent microbial infection effectivelyor which allow an efficient treatment thereof.

In an preferred embodiment of the invention said prophylaxis or therapyis a prophylaxis or therapy for the treatment, prevention oramelioration of an infection.

In an even more preferred embodiment of the invention said infection isselected from the group of fungal, viral, protozoal, parasitical andbacterial infections.

In a further preferred embodiment of the present invention the infectionis selected from the group consisting of pneumonia, invasive fungalinfection, enterocolitis, soft-tissue infection, and sepsis.

Said medical treatments may comprise the administration ofantimicrobials or other substances, such as ciprofloxacin, amphotericinB, fluconazole, trimethoprim-sulfamethoxazole, acyclovir,piperacillin/tazobactam, gentamicin, meropenem, vancomycin or anycombination administered simultaneously or subsequent of saidantimicrobials or substances. Many other antimicrobials or substancescould be administered. Also, cell preparations like granulocytetransfusions or others could be given. The dosage and schedule of suchantimicrobials or other medical treatments depend on whether prophylaxisor therapy of microbial infections is to be achieved. Moreover, aclinician must also consider what infectious agents s/he has to expectand what infectious agents are isolated from the patient. A criticalvalue for a suitable antimicrobial prophylaxis or therapy is thecapability of the subject for hematopoietic recovery. Theseconsiderations have been made so far based on empirical data. Thanks tothe present invention, the hematopoietic cytocapacity, as a criticallandmark for hematopoietic recovery and the risk of infection in asubject, can be determined and allow to select a suitable prophylaxisand therapy without merely relying on empiric and average data.

Further, the present invention relates to a method for selecting asuitable prophylaxis or therapy for neutropenic fever for a subject,wherein said method comprises the steps of the aforementioned method andthe further step (c) selecting a suitable prophylaxis or therapy forneutropenic fever based on the results obtained in step (b).

The definitions and explanations of the terms made above apply mutatismutandis.

The term “suitable prophylaxis or therapy for neutropenic fever”encompasses medical treatments which either prevent neutropenic fevereffectively or which allow an efficient treatment thereof. Said medicaltreatments may use oral or intravenous administration of single orcombinations of antimicrobials or biological humoral or cellulartherapies. Preferably, a suitable prophylaxis or therapy for neutropenicfever involves administration of recombinant growth and developmentfactors or cytokines like G-CSF and others as single or combinationtreatment in different dosage and schedule. Most preferably, saidrecombinant growth and development factors or cytokines are administeredprophylactically. A critical value for a suitable prophylaxis or therapyof neutropenic fever is the capability of the subject for hematopoieticrecovery. These considerations have been made so far based on empiricaldata.

Thanks to the present invention, the hematopoietic cytocapacity, as acritical landmark for hematopoietic recovery and the risk of infectionin a subject, can be determined and allow to select a suitableprophylaxis and therapy of neutropenic fever without merely relying onempiric and average data.

Furthermore, the present invention relates to a method for selecting asuitable amount of hematopoietic stem cells, preferably CD34⁺ cells tobe transfused for the therapy of a subject, wherein said methodcomprises the steps of the method of claim 1 and the further step (c) ofselecting the amount of said cells to be transfused for the therapy of asubject based on the results obtained in step (b).

The definitions and explanations of the terms made above apply mutatismutandis.

The term “a suitable amount of hematopoietic stem cells, preferablyCD34⁺ cells for the therapy” refers to an amount of said cells whichallows efficient hematopoietic recovery.

Also, the present invention relates to a method for selecting a suitableamount of a hematopoietic growth factor or cytokine for the treatment ofa subject, wherein said method comprises the steps of the aforementionedmethod and the further step (c) selecting a suitable amount of ahematopoietic growth factor or cytokine for the treatment of saidsubject based on the results obtained in step (b).

The definitions and explanations of the terms made above apply mutatismutandis. The term “a suitable amount of a hematopoietic growth factoror cytokine” refers to an amount of a hematopoietic growth factor whichsupports efficient hematopoietic recovery. Preferably, saidhematopoietic growth factor is G-CSF, or other factors like GM-CSF,erythropoietin, stem cell factor, thrombopoietin or KGF. Morepreferably, a suitable dose of G-CSF is about 5 μg/kg per day appliedsubcutaneously.

The methods of the present invention encompass in vitro applications.

The present invention also comprises a method for determining thehematopoietic cytocapacity of a subject comprising the steps of:

-   -   (c) determining a parameter, preferably a parameter of a sample,        preferably a body fluid, preferably blood or a blood derived        sample, wherein the parameter is preferably a marker of a        population or sub-population of cells, preferably hematopoietic        cells, in said sample obtained from a mammal, wherein said        mammal has been subjected to administration of a single dose of        a hematopoietic growth factor, preferably G-CSF, and has been        maintained for a time sufficient to allow mobilization or        release of leukocytes from hematopoietic production and storage        tissues and sites of margination into the blood; and    -   (d) determining the hematopoietic cytocapacity by assessing the        value of the parameter determined in step (a) with the value of        the parameter measured in a like manner in a control mammal        wherein said control mammal is selected from the group        consisting of mammals having (i) a high risk for a disease,        disorder or complication associated with high-dose cytotoxic        chemotherapy and/or hematopoietic cell transplantation, (ii) an        intermediate risk for a disease, disorder or complication        associated with high-dose cytotoxic chemotherapy and/or        hematopoietic cell transplantation or (iii) a low risk for a        disease, disorder or complication associated with high-dose        cytotoxic chemotherapy and/or hematopoietic cell        transplantation.

Even more preferred said mammal is a human. Further preferred, saidmarker is a T cell associated marker, or a B cell associated marker.Markers within the ambit of the present invention are specified, e.g, inExample 7.

Finally, the present invention encompasses a method of treating adisease, disorder or complication associated with high-dose cytotoxicchemotherapy and/or hematopoietic cell transplantation or asusceptibility therefor in a subject as specified herein comprising thesteps of the methods specified herein and the further step ofadministering to said patient (i) a suitable antimicrobial prophylacticor therapeutic agent in an effective amount, (ii) a suitable agent forthe prophylaxis or therapy of neutropenic fever in an effective amount,(iii) an effective amount of hematopoietic stem cells, preferably CD34⁺cells, or (iv) an effective amount of a hematopoietic growth factor.

The definitions and explanations of the terms made above apply mutatismutandis. The dosage regimen for said administration of an agent, cellsor growth factor will be determined by the attending physician and otherclinical factors; preferably in accordance with any one of the abovedescribed methods. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Progress can be monitored by periodicassessment.

Encompassed by the present invention is the use of leukocytes obtainedfrom a subject for the preparation of a diagnostic composition fordiagnosing a susceptibility for a disease, disorder or complicationassociated with high-dose cytotoxic chemotherapy and/or hematopoieticcell transplantation in said subject, wherein said subject has beensubjected to administration of a dose of G-CSF and has been maintainedfor a time sufficient to allow mobilization or release of the leukocytesfrom hematopoietic production and storage tissues and sites ofmargination into the blood.

The definitions and explanations of the terms made above apply mutatismutandis.

The term “diagnostic composition” as used in accordance with thisinvention refers to an entity of the leukocytes obtained from the saidsubject which allow determination of a therapeutic or prognostic valueof said leukocytes, preferably their absolute or relative number in agiven volume of blood. Dependent on the method of determination of thenumber of leukocytes, said cells may be pretreated, e.g., forfluorescence activated cell sorting (FACS) analysis or may be applied toa suitable counting device such as a counting slide. Such pretreatmentmeasures are well known in the art.

In light with the foregoing, in a preferred embodiment of the method orthe use of the invention said disease, disorder or complicationassociated with high-dose cytotoxic chemotherapy and/or hematopoieticcell transplantation, is neutropenic fever, microbial infection, delayedhematopoietic recovery, bleeding, immunosuppression, immunologicaleffects directed against the host, high level of supportive care,morbidity and mortality.

In another preferred embodiment of the method or the use of theinvention said subject is a human.

In a furthermore preferred embodiment of the method or the use of theinvention said subject has been subjected to high-dose chemotherapy.

The term “high-dose chemotherapy” refers to the dosage and intensity ofchemotherapy which is regarded as “high-dose” when a significantmyelosuppression follows with significantly reduced peripheral bloodleukocyte and platelet counts and an increased risk of infection.Depending on the dosage of the chemotherapeutic substances, atransplantation of PBSCs may be required or not. Frequently, G-CSF orGM-CSF are applied after high-dose chemotherapy and transfusions of redblood cells or platelets are required.

Most preferably, said high-dose chemotherapy comprises administration ofmelphalan, busulfan, cyclophosphamide, carmustine, etoposide andcytarabine or other chemotherapeutic substances. In the high-dosechemotherapy, escalated dosages can be applied. High-dose regimens canconsist of a combination of substances in escalated dosages withsubstances in conventional dosages. For additional immunosuppression inhigh-dose chemotherapy anti-thymocyte globulin (ATG) or anti-lymphocyteglobulin (ALG) could be included in the regimens.

In another more preferred embodiment of the method or the use of theinvention said subject has been subjected to myelosupressivechemotherapy.

The term “myelosupressive chemotherapy” refers to the effect of thechemotherapy on the bone marrow functions. A chemotherapy ismyelosuppressive when the peripheral blood leukocyte counts and possiblyplatelet counts decrease for a limited period of time and recoverthereafter spontaneously or under hematopoietic growth factorstimulation. Depending on the degree of myelosuppression there can be anincreased risk of infection.

Most preferably, said myelosupressive therapy comprises theadministration of cyclophosphamide, etoposide, carmustine, cytarabine,melphalan, busulfan, doxorubicin, epirubicin, paclitaxel, docetaxel,thiotepa, fludarabine, vincristine, bendamustine, cisplatin,carboplatin, daunorubicin, fluorouracil, gemcitabine, idarubicin,ifosfamide, irinotecan, methotrexate, mitoxantrone, oxaliplatin,treosulfan, vinblastine, vinorelbine. Other chemotherapeutic substancescould be administered as well.

In a furthermore preferred embodiment of the method or the use of theinvention said subject has been subjected to radiotherapy, suffers froma primary or secondary bone marrow disease, an autoimmune disease, ahereditary disease or disorder or an infection.

The term “radiotherapy” refers to any therapeutic application of ionicradiation. Said radiation may be radioactive radiation including fastelectrons, neutrons, protons, or Pi-mesons, microwaves, IR, and UVradiation. Preferably, said radiation is used to treat cancer ormalignant hematopoietic diseases.

The term “primary bone marrow disease” encompasses those diseases anddisorders which primarily effect the cells of the bone marrow. Examplesfor said diseases or disorders are acute or chronic leukemias,myelodysplasia, aplastic anemia, congenital neutropenia, cyclicneutropenia, idiopathic and autoimmune neutropenia.

The term “secondary bone marrow disease” encompasses those diseases anddisorders where the bone marrow becomes involved secondarily like incase of bone marrow metastases of a cancer.

The term “autoimmune disease” refers to those diseases which areassociated with the presence of autoantibodies in a patient. Examplesfor said diseases or disorders are rheumatoid arthritis or lupuserythematosus.

The term “hereditary disease or disorder” encompasses all diseases ordisorders which are caused by genetic defects that will be transmittedvia the germ line. Most preferably, said hereditary disease or disorderwithin the scope of this invention is cyclic neutropenia, Kostmannsyndrome, Shwachman syndrome and Gaucher's disease.

The term “infection” preferably encompasses those infections which canresult in life-threatening conditions for the subject. Infections canlead to serious, organ dysfunction and organ failure in themyelosuppressed host. Such infections can be caused by bacteria,viruses, fungi, protozoa and parasites like staphylococci, streptococci,enterococci, escherichia coli, klebsiella species, Pseudomonasaeruginosa, candida species, Aspergillus species, Pneumocystis carinii,cytomegalovirus, herpes viruses, respiratory viruses and many othermicrobials. It is understood by the person of skill in the art that witha myelosuppressed patient, any infection may potentially belife-threatening. However, it is further to be understood that by thepresent invention there are also contemplated embodiments where theinfection that is to be treated, ameliorated and/or prevented may not belife-threatening or may not lead to life-threatening conditions.Nevertheless, application of the cytocapacity test in a subject may leadthe attending physician to consider treatment, prevention and/oramelioration of any such infection.

In accordance with the aforesaid, the invention provides a method fordetermining the hematopoietic cytocapacity of a subject comprising thesteps of:

-   -   (a) determining the amount of leukocytes present in a blood        sample obtained from a subject, wherein said subject has been        subjected to administration of a single dose of G-CSF and has        been maintained for a time sufficient to allow mobilization or        release of the leukocytes from hematopoietic production and        storage tissues and sites of margination into the blood; and    -   (b) determining the hematopoietic cytocapacity by assessing the        amount of leukocytes determined in step (a) with the amount of        leukocytes which have been mobilized or released in a control        subject.

The invention preferably provides the above method further comprising astep (c) wherein (c) comprises selecting a suitable antimicrobialprophylaxis or therapy, or hematopoietic growth factor treatment, forsaid subject based on the results obtained in step (b).

The above control subject may be a healthy control subject. Preferably,the control subject is selected from the group consisting of subjectshaving (i) a high risk for a disease, disorder or complicationassociated with high-dose cytotoxic chemotherapy and/or hematopoieticcell transplantation, (ii) an intermediate risk for a disease, disorderor complication associated with high-dose cytotoxic chemotherapy and/orhematopoietic cell transplantation or (iii) a low risk for a disease,disorder or complication associated with high-dose cytotoxicchemotherapy and/or hematopoietic cell transplantation.

In another more preferred embodiment of the method or the use of theinvention said G-CSF is filgrastim (Neupogen™; Amgen Inc., ThousandOaks, Calif., USA) or lenograstim (Granocyte™; Chugai, Japan).

Said G-CSF preparations are commercially available and have beenapproved from the drug regulation administrations for the purpose ofmedical treatments. Thus, application of said preparations is preferredfor the methods and uses of this invention.

Preferably, said dose of G-CSF is selected from a range of about 1 toabout 100 μg/kg body weight of the said subject. More preferably, thedose of G-CSF is selected from a range of about 1 to about 20 μg/kg bodyweight of the said subject.

Also more preferably, said dose of G-CSF is 1.0, 2.5, 5, 7.5, 10 or 15μg/kg body weight of the said subject

Further preferably, the dose of G-CSF is selected from a range of about15 to about 30, 60 and about 100 μg/kg body weight of the said subject.Still further preferably, the dose of G-CSF is selected from a range ofabout 20 to about 30, 60 and about 100 μg/kg body weight of the saidsubject. Without being bound by theory, it is the belief of theinventors that higher doses of G-CSF (about 15 μg/kg body weight of thesaid subject or more) lead to more pronounced differentiation of thepatients and therefore may improve the predictive value of thecytocapacity test. This may especially be true for patients that havenot undergone high-dose treatment or for healthy subjects. Ifalternative dosing, e.g., based on the body surface area is used,similar absolute amounts of G-CSF are intended.

In a more preferred embodiment of the method or the use of the inventionsaid time sufficient to allow mobilization or release of the leukocytesis in the range of 1 to 120 hours.

Also more preferably, said time sufficient to allow mobilization orrelease of the leukocytes is at least 1 hour, at least 2 hours, at least6 hours, at least 10 hours, at least 12 hours, at least 14 hours or atleast 18 hours.

Several documents are cited throughout the text of this specification.Full bibliographic citations may be found at the end of thespecification immediately preceding the claims. Each of the documentscited herein (including any manufacturer's specifications, instructions,etc.) is hereby incorporated by reference.

The Figures show:

FIG. 1: Leukocyte peak after single dose G-CSF and leukocyte course. TheG-CSF-induced leukocyte peak was observed (day 0) in a patient withmultiple myeloma of this seris who received high-dose melphalantreatment. Autologous PBSC transplantation was performed approximately 1hour after the leukocyte peak measurement. HDT, high-dose chemotherapy.

FIG. 2: The independence of the cytocapacity test from the CD34⁺ cellnumber in the prediction of the neutrophil and platelet recovery. Thekinetics of the neutrophil (absolute neutrophil count, ANC) (left) andplatelet (PLT) recovery (right) are shown for separated CD34⁺ celllevels. Within the respective CD34⁺ cell level, a stratification for thecytocapacity test (≦median, >median) was performed. A stratifiedlog-rank test was used to investigate the independent prediction of thehematopoietic recovery by the cytocapacity test over the three indicatedCD34⁺ cell levels.

FIG. 3: Prediction of fever >38.0° C. and of antimicrobial therapy bythe cytocapacity test. The categories of the cytocapacity test are basedon the established relative scale where the median of the leukocyte peakdistribution was set as 1.0. The proportion of cases within thecategories without fever >38.0° C. (A) and the corresponding median daysof intravenous antimicrobial therapy (B) are presented. On the leftside, the data is shown for the entire number of cases, on the rightside cases which had received a CD34⁺ cell number ≦2.5×10⁶/kg wereexcluded.

FIG. 4: The release of the G-CSF reaction. A single dose of G-CSF (5μg/kg) was given approximately 30 hours after the completion ofhigh-dose chemotherapy. The induced leukocyte peak which is shown for 5different patients was measured after 12-14 hours. The autologous bloodstem cell transplantation was performed afterwards.

FIG. 5: Distribution of WBC peaks representing the G-CSF reactionordered according to magnitude (A) and correlation of the G-CSF reactionwith the rate of documented infections (B).

The present invention is illustrated by reference to the followingbiological Examples which are merely illustrative and are not to beconstructed as a limitation of the scope of the present invention.

EXAMPLE 1 The Cytocapacity Test

Patients

Eighty-seven patients with multiple myeloma (MM) and relapsednon-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) receivedhigh-dose therapy with standard regimens (Philip 1995; Barlogie 1997;Weaver 1998; Linch 1993, Palumbo 1999) followed by a single dose ofG-CSF before autologous peripheral blood stem cell (PBSC)transplantation at one institution. The patients gave informed consent.The characteristics of the patients are presented in table 1.

PBSC Mobilization, Harvesting, Processing and Cryopreservation

In the vast majority of patients, the IEV regimen with G-CSF was usedfor mobilization. The IEV regimen consists of ifosfamide 2500 mg/m²intravenously day 1-3, epirubicin 100 mg/m² intravenously day 1 andetoposide 150 mg/m² intravenously day 1-3 followed by G-CSF (filgrastim;Amgen, Thousand Oaks, Calif., USA) at a dose of 5 μg/kg subcutaneouslydaily from day 5 until the completion of PBSC harvesting. Apart fromindividual dose reductions, patients with MM at an age ≧60 yearsreceived IEV in a 75% dosage since 2000. PBSCs were harvested when thepost-nadir, G-CSF stimulated leukocyte count rose up to 5000-10000/μL orabove using a COBE Spectra (COBE, Heimstetten, Germany) or an AS104(Fresenius, St. Wendel, Germany) cell separator and standard programs.In 20 patients (23%) with MM or low-grade NHL, harvested PBSC from asingle leukapheresis underwent immunomagnetic B cell purging (MaxSep,Baxter Immunotherapy, Unterschleissheim, Germany) immediately after thecollection. The PBSCs were mixed with an equal volume of a freezingsolution which was prepared with 5% HSA and 100% DMSO (Cryoserv, TeraPharmaceuticals, Midvale, Utah, USA) (4:1). The final DMSO concentrationwas 10%. After computerized controlled-rate freezing, the bagscontaining the PBSCs were stored in the vapor phase of liquid nitrogen.

CD34⁺ Cell Enumeration and PBSC Dose

The determination of CD34⁺ cells was carried out according to theguidelines of ISHAGE (Sutherland 1993) using a FACScan (BD, MountainView, Calif., USA) or an EPICS XL-MCL (Electronics, Miami, Fla., USA)flow cytometer equipped with an argon laser. Whole blood was incubatedfor 30 minutes at 4° C. in the dark with the PE conjugated monoclonalanti-CD34 antibody and the FITC conjugated monoclonal anti-CD45 antibodyfollowed by a wash and red blood cell lysis (BD). Before 1998, 20000cells were analysed, 75000 cells thereafter. To exclude cell debris,platelets, remaining red cells and all CD45 negative cells, a forwardscatter versus CD45 fluorescence dot plot was used. The double-positiveCD34⁺/CD45⁺ cell population was then defined and backgated for low CD45expression and low side scatter properties. The percentage of the sodefined CD34⁺ cells was multiplied with the total nucleated cell contentof the apheresis product to result in the absolute number of CD34⁺ cellsharvested. The nucleated cell content was determined by automated cellcounting using a Coulter STKS (Coulter, Miami, Fla., USA). The anti-CD34antibody (HPCA-2), the anti-CD45 antibody (2D1) and the isotype controlsused were from BD.

High-dose Therapy and Autotransplantation

Fifty-eight patients with MM (67%) were either treated according to the“Total Therapy” concept of Barlogie (Barlogie 1997) and were to receivetandem melphalan 200 mg/m² (MEL200) within 3-6 months or at an agebetween 60 and 70 years were treated with tandem melphalan 100 mg/m²(MEL100) according to Palumbo (Palumbo 1999). The younger patients withMM who did not reach a partial remission after a first melphalan 200mg/m² were to receive busulfan 12-16 mg/kg and cyclophosphamide 120mg/kg as second high-dose treatment (BUCY). Twenty-nine patients withrelapsed NHL or HD (33%) were treated either with busulfan 16 mg/kg andcyclophosphamide 120 mg/kg (BUCY) or with carmustine 300 mg/m²,etoposide 800 mg/m², cytarabine 800 mg/m² and cyclophosphamide 140 mg/kg(BEAC) (Philip 1995) or with carmustine 300 mg/m², etoposide 800 mg/m²,cytarabine 1600 mg/m² and melphalan 140 mg/m² (BEAM) (Linch 1993). Thedistribution of high-dose regimens is shown in table 1. Autologous PBSCtransplantation was performed 48-60 hours after the last dose ofchemotherapy. The PBSC products were thawed in a 37° C. water bath atthe bedside and were reinfused through a central venous catheter afterthe addition of 20 mL ACD-A.

Application of G-CSF and Blood Cell Counts

G-CSF was always given more than 24 hours after the last chemotherapyinfusion, as recommended (Ozer 2000). The recombinant human G-CSF givensubcutaneously after high-dose chemotherapy was filgrastim (Amgen,Thousand Oaks, Calif., USA) in 113 cases and lenograstim (Chugai, Japan)in 9 cases. The single G-CSF injection evaluated was given on theevening before PBSC autograft at a dose of 5 μg/kg. The inducedleukocyte peaks were detected with the routine blood tests on the nextmorning, approximately 14 hours after the single G-CSF dose. Thetransplantation of autologous PBSCs was carried out approximately 1 hourafter the routine blood test. From the day after PBSC transplantation,G-CSF was given daily at a dose of 5 μg/kg until leukocyte counts werebetween 5000/μL and 10000/μL following aplasia. Routine blood cellcounts including a differential were performed on a Coulter STKS(Coulter, Miami, Fla., USA) daily from the start of high-dose therapyuntil the patients left the hospital.

Supportive Care

All the patients were hospitalized during high-dose therapy, autograftand the post-transplant phase. The antimicrobial prophylaxis consistedof the following: From the time of autograft until neutrophil recoveryciprofloxacin 2×250 mg daily was given. Oral amphotericin B suspension4×1 mL was given during the entire hospital stay or, alternatively, incase of oral amphotericin intolerance, oral fluconazole 1×100 mg daily.Pneumocystis carini pneumonitis prophylaxis was started during theadministration of high-dose chemotherapy withtrimethoprim/sulfamethoxazole 160 mg/800 mg 2×1 daily and was continuedafter neutrophil recovery with 2×1 on two consecutive days per week for6 months. In case of trimethoprim/sulfamethoxazole intolerance,alternatively a pentamidin inhalation with 300 mg was performed every 4weeks for 6 months from autograft. Intravenous acyclovir was given at adose of 2×500 mg from the start of high-dose therapy until neutrophilrecovery. Red blood cell products and single-donor platelets weresubstituted to maintain a hemoglobin level above 80 g/L and a plateletcount above 10000/μL. All blood cell products were CMV negative, wereirradiated with 30 Gy and were transfused through a leukocyte reductionfilter. Empirical intravenous antimicrobial therapy was started duringneutropenia after a single oral temperature >38.5° C. or whenfever >38.0° C. was present over at least one hour and was carried outaccording to previously published guidelines (Hughes 1997). The initialtreatment was carried out with piperacillin/tazobactam plus gentamicin.Escalation was performed with meropenem/vancomycin. If fever persistedon days 5-7, intravenous amphotericin B was added. The antimicrobialtreatment was continued until neutrophil recovery and at least 24 hoursafter the resolution of fever. Patients were discharged from thehospital after neutrophil and platelet recovery and after the cessationof antimicrobial therapy.

Assessment of Hematopoietic Recovery, Fever and Supportive Care

Neutrophil recovery was defined as the first day with a neutrophil countabove 500/μL from the day of autograft. Platelet recovery was defined asthe first day with an unsubstituted platelet count above 20000/μL fromthe day of autograft. The occurrence of fever >38.0° C., the number ofdays with fever >38.0° C. and the days with intravenous antimicrobialtherapy were recorded until the patients were discharged from thehospital.

Objectives and Statistical Analysis

The objectives of this investigation were to correlate the G-CSF-inducedleukocyte peak with the neutrophil and platelet recovery, the incidenceof neutropenic fever and the duration of intravenous antimicrobialtherapy and to compare these correlations with those obtained for theCD34⁺ cell content of the PBSC autograft. All analyses presented (exceptfor the demographic and disease baseline characteristics) are based ontransplantation courses as units of observation.

The rates of neutrophil and platelet recovery over time were estimatedusing the product-limit method according to Kaplan and Meier (1958) andprognostic subgroups were compared by use of the log-rank test (Peto1972). Differences in continuous variables between prognostic groupswere tested by Wilcoxon-Mann-Whitney or Kruskal-Wallis analysis,depending on the number of groups. In case of nominal or orderedcategories, Fisher's exact or chi² tests for trend were applied.Multivariate analysis was carried out with stratified versions of therespective tests or Cox models (Cox 1972) in order to examine theindependent impact of the predictive factors with regard to thedifferent end-points. All reported p-values result from two-sided tests.

Performance of the Cytocapacity Test

A single subcutaneous G-CSF injection (5 μg/kg) was investigatedfollowing 122 high-dose chemotherapy courses in 87 patients withmultiple myeloma or lymphoma. The induced peripheral blood leukocytepeaks were detected with the routine blood tests approximately 14 hourslater. These leukocyte peaks (FIG. 1) consisted of around 90%neutrophils. The patient and treatment characteristics are presented intable 1, the induced leukocyte peaks, the neutrophil and plateletrecovery, the absence of fever >38.0° C. and the requirements forintravenous antimicrobial therapy in table 2. The time interval betweenthe start of intravenous chemotherapy and the single G-CSF injection hadan effect on the median of the induced leukocyte peaks. The oraladministration of busulfan for 4 days in the BUCY regimen, however,which preceded the 2 day-intravenous administration of cyclophophamide,did not reduce the leukocyte peaks compared to the MEL regimen with a 2day-intravenous administration of melphalan.

In case of BUCY and MEL high-dose therapy, with a 2.5 day-intervalbetween the start of intravenous chemotherapy and single dose G-CSF, themedian leukocyte peak was 16200/μL. In case of BEAM and BEAC high-dosetherapy, with a 6.5 day-interval, the median leukocyte peak was 4100/μL,which was a factor 4 lower. The observed objectives of this analysis,however, were comparable between the BUCY/MEL and the BEAC/BEAM group(table 2). This suggested to evaluate the leukocyte peaks on a relativescale which was defined by the median (set as 1.0) of the distributionand was applied on the BUCY/MEL and the BEAC/BEAM group. For statisticalanalysis, categorizations of the leukocyte peak values in below (≦50.percentile) and above (>50. percentile) the median and subsequently into≦25. percentile, >25.-≦50. percentile and >50. percentile were used. Theentire median-orientated values on the relative scale corresponding tothe minimum, 25. percentile, 50. percentile and maximum were 0.15, 0.77,1.0 and 3.66, respectively. The test is termed cytocapacity test.

EXAMPLE 2 Prediction of the Hematopoietic Recovery

The median time to neutrophil (>500/μL) and platelet (>20000/μL)recovery was 9 and 10 days, respectively. The cytocapacity testpredicted the neutrophil (p=0.001) and the platelet recovery (p<0.0001)(table 3). Interestingly, for the leukocyte counts before the test, aborderline significant correlation with the neutrophil recovery (p=0.06)but no correlation with the platelet recovery (p=0.4) was found. Thenumber of CD34⁺ cells in the autologous PBSC graft also predicted theneutrophil (p<0.0001) and platelet recovery (p<0.0001) (table 3), asexpected.

The cytocapacity test itself was independent from the PBSC autograftsince the test was performed before the cryopreserved autologous PBSCswere transfused. The correlation between the induced leukocyte peaks andthe CD34⁺ cell number was weak (r=0.226; p=0.01). In multivariateanalysis, performed as a stratified log-rank test, the independentimpact of the cytocapacity test (<=median, >median) in the prediction ofthe neutrophil (p=0.03) and the platelet recovery (p=0.01) could befollowed through the different CD34 levels, increased with a lower CD34⁺cell number and modified the CD34⁺ cell dose effect of the PBSCautograft. In a Cox model where the CD34⁺ cell number of the autograftwas included as a quantitative variable, the independent impact of thecytocapacity test on the neutrophil (p=0.05) and platelet recovery(p=0.0007) was confirmed.

EXAMPLE 3 Determination of a Favorable CD34⁺ Cell Threshold forTransplantation

The cytocapacity test itself was independent from PBSC transplantationsince the test was performed before the transfusion of the cryopreservedPBSCs. In multivariate analysis, the cytocapacity test independentlyfrom the dose effect of the PBSC CD34⁺ cells predicted the hematopoieticrecovery. This could have practical consequences for choosing athreshold dose of CD34⁺ cells for transplantation. With a cytocapacitytest >1.0, the transplantation of >2.5×10⁶ CD34⁺ cells/kg was sufficientto achieve a favorable neutrophil and platelet recovery, completed in 10and 12 days, respectively. In cases with a cytocapacity test <1.0, theincreased risk for a delayed hematopoietic recovery would suggest to usea higher number of CD34⁺ cells.

EXAMPLE 4 Prediction of Neutropenic Fever and Intravenous AntimicrobialTherapy

Fever >38.0° C. was absent in 55 of 122 procedures (45%). The mediannumber of days with fever >38.0° C. was one day and intravenousantimicrobial therapy was given for a median of 4 days. The cytocapacitytest was correlated with the absence of fever (p=0.03) and the medianduration of intravenous antimicrobial therapy (p=0.03) (table 3). Thesearch for a correlation of the. CD34⁺ cell number in the PBSC autograftwith the absence of fever >38.0° C. showed a trend (p=0.07) but nocorrelation with intravenous antimicrobial therapy was found (p=0.3).Both the cytocapacity test and the CD34⁺ cell number correlated with theduration of leukopenia <1000/μL (p<0.0001) whereas only the cytocapacitytest correlated with the severity of leukopenia (p=0.02). A continuousdirect relation between the cytocapacity test and the absence of feverand an inverse relation with the requirements for intravenousantimicrobial therapy was observed (FIG. 3). For the subgroup with a lowcytocapacity test <0.6, the absence of fever was only 21% whereas forthe subgroup with a high cytocapacity test >2.0 the absence of fever was80%. The exclusion of cases which had received a PBSC autograft with alow CD34⁺ cell number (<=2.5×10⁶/kg) and which therefore represent anunfavorable group from the analysis did not change this correlation(FIG. 3). Three treatment-related deaths occurred. These were caused bysepsis. The cytocapacity test in the three treatment-related deaths was0.15, 0.77 and 1.0.

EXAMPLE 5 The Possibility of Outpatient Care

The fast hematopoietic recovery with the use of mobilized PBSCs afterhigh-dose chemotherapy and the associated good tolerability and lowtreatment-related mortality below 5% has spurred the interest inperforming autografts on an outpatient basis. With a cytocapacitytest >1.0 in combination with a standard dose of CD34⁺ cells(>2.5×10⁶/kg), outpatient care as a possibility was suggested. Thisconstellation was associated with an optimal hematopoietic recovery(FIG. 2) and a reduced risk of infection (FIG. 3).

EXAMPLE 6 Factors Influencing Cytocapacity Test and CD34⁺ Cell Number

For age, gender, diagnosis and pretreatment with chemotherapy andradiotherapy, no significant correlation with the cytocapacity test orthe CD34⁺ cell number in the PBSC autografts was found. TABLE 1Characteristics of The Patients And Specification of High-doseChemotherapy And Peripheral Blood Stem Cell (PBSC) Autograft. PatientsNo. 87 100%  Age (years) Median 53 Range 18-68 Gender Female 36 41% Male51% 59% Disease Hodgkin's Diseas 8  9% Non-Hodgkin's Lymphoma 21 24%Multiple Myeloma 58 67% Previous Chemotherapy (cycles) Median 7 Range0-25 Previous Radiotherapy Yes 32 37% No 55 63% High-dose chemotherapyNo. 122 100% BEAM 5  4% BEAC 8  7% BUCY 22 18% MEL 87 71% PBSC AutograftFirst 78 64% Second 41 33% Third 2  2% Fourth 1  1%

TABLE 2 Leukocyte Counts In The Blood Before And After The Single G-CSFDose, The Duration Of Leukopenia <1000/μL And The Objectives Of TheInvestigation. The recovery of the absolute neutrophil count (ANC) andthe platelet (PLT) recovery, the absence of fever >38.0° C. and theduration of intravenous (i.v.) antimicrobial therapy were the objectivesof the investigation. BUCY/ BEAC/ High-dose chemotherapy All MEL BEAMNo. of procedures 122 Maximum 1100 1100 400 Leukopenia <1000/μL (days)Minimum 3 3 6 50. percentile 6 5 7 Maximum 11 11 10 Days to ANC >500/μL

Minimum 8 8 8 25. percentile 8 8 8 50. percentile 9 9 8 75. percentile10 10 9 Maximum 12 12 11 Days to PLT >20000/μL Minimum 8 8 8 25.percentile 10 10 10 50. percentile 10 10 10 75. percentile 11 11 12Maximum 24 23+ 24 Fever >38.0° C. No fever 45% 45% 46% i.v.Antimicrobials (days) Minimum 0 0 0 50. percentile 4 4 4 Maximum 28 2817

TABLE 3 Correlation of the cytocapacity test and the CD34⁺ cell numberof the PBSC autograft with the objectives of the investigation. CD34⁺ ×10⁶/kg Cytocapacity test ≦2.5 >2.5-≦5.0 >5.0 P Value ≦25. >25.-≦50. >50.percentile P Value No. of procedures 30 40 52 32 31 59 Days to ANC >500/μL <0.0001 0.001 Minimum  8  8  8  8  8  8 25. percentile 10  9  8 9  8  8 50. percentile 10  9  8 10  9  9 75. percentile 10 10  9 10 10 9 Maximum 12 11 10 12 12 11 Days to PLT > 20000/μL <0.0001 <0.0001Minimum  9  9  8  9  8  8 25. percentile 11 10  9 10 10  9 50.percentile 12 11 10 12 10 10 75. percentile 13 12 11 15 11 11 Maximum 17+ 24  23+ 24  14+ 14 Fever > 38.0° C. 0.7 0.03 No fever    33%    43%   54%    28%    48%    53% i.v. Antimicrobials (days) 0.3 0.03 Minimum 0  0  0  0  0  0 25. percentile  0  0  0  2  0  0 50. percentile  5  3 5  6  5  2 75. percentile  9  6  7 10  7  6 Maximum 13 17 28 17 28 17

EXAMPLE 7 Factors That Correlate with Hematopoietic Recovery

The cytocapacity test according as described hereinabove involves themeasurement of parameters of a mammal or of parameters derived from asample derived from a mammal which correlate with the hematopoieticrecovery where the mammal has been subjected to administration of ahematopoietic growth factor. The parameter is preferably white bloodcell count, or leukocyte cell count. The absolute values of theparameter measured is, as described above, preferably evaluated on arelative scale which is defined by the median (set as 1.0) of thedistribution within a treatment group, that is, within a group ofmammals receiving a similar or identical treatment. The term “similar oridentical treatment” preferably relates to the treatment regimencomprising the doses of radiation therapy and chemotherapy, and thetimes of administration and kinds of drugs used therein. The parametersare preferably measured in a sample derived from the mammal, such as abody fluid or a biopsy. The biopsy is preferably taken from a site thatcontains, in a healthy mammal, immunologically active cells. The cellsare preferably T cells, B cells, Granulocytes, Platelets, Monocytes, NKcells, and the like cells. The cells may be derived from an early orlate stage in cell development and/or differentiation. The body fluid ispreferably blood or derived from blood. The mammal is preferably ahuman. The growth factor is preferably G-CSF or a growth factor ofsimilar activity in stimulating the hematopoietic system. For instance,angiotensin and/or angiotensin-derived peptides (Rodgers et al., CancerChemother Pharmacol 49(5):403-11, 2002), interleukin-1 beta (Lebedev etal., Radiats Biol Radioecol 42(1):60-4, 2002), interleukin-8 (Terashimaet al., Blood 92(3):1062-69, 1998; Laterveer. et al., Blood85(8):2269-75, 1995; Laterveer et al., Blood 87(2):781-88, 1996) andinterleukin-11 (Saitoh et al., Cytokine 13(5):287-94 2001), may exhibitsuch activity. The stimulating activity on the hematopoietic system mayvary from one factor to the other. For instance, interleukin-11preferably acts as on megakaryopoiesis. Also non-peptide factors, suchas PGG-glucan, may act in stimulating hematopoiesis (Turnbull et al.,Acta Haematol 102(2):66-71, 1999).

The factors that may be measured include cell count. This is preferablymeasured in a sample derived from the mammal, preferably a human, thesample being preferably a blood sample or blood-derived sample. Theblood derived sample preferably contains white blood cells, alsopreferably leukocytes. The cells counted are preferably T cells, NKcells, Granulocytes, eosinophilic cells, Monocytes, Neutrophils,thrombocytes, lymphocytes, B cells, and the like cells. The cellscounted preferably are of hematopoietic origin, more preferably cellsthat are derived from precursor and/or stem cells.

The parameter is also possibly derived from measurement of a marker,preferably a cell surface marker. Such markers include CD3, CD4, CD8,CD20, B7, CD45, CD34, CD36, CD56, CD19, CD20-24, CD25, CD 37, CD79 alphaand/or beta, CD 2, CD5, CD7, CD43, CD45 (leukocyte common antigen LAK)CD45RO, CD56, flow cytometry side scatter, flow cytometry forwardscatter, S-100 markers which react with all lymphoid cells, CD30,CD45RA, CD74, CDw75, CDw76, CD79, kappa light chain, lambda light chain.Additional markers are those that are associated with cellproliferation, for instance, the Ki-67 marker. Further markers areassociated with cell death, preferably apoptosis or necrosis. A numberof markers or methods are known to the person skilled in the art for thedetection of apoptosis. For instance, annexin V, DNA laddering, nucleuscondensation, nucleus fragmentation, single strand DNA labeling,cytochrome c release and the cleavage of substrates (e.g. PARP) bycaspases are markers associated with apoptosis. Preferably, annexinV-labeling or caspase3/7 activity test can be employed.

The determination and/or measurement of the above parameters is wellwithin the knowledge of the person of skill in the art. For instance,assays for the surface and cell internal protein markers mentioned aboveare available commercially from Dako Cytomation Denmark A/S,Produktionsvej 42, 2600 Glostrup, Denmark, or a subsidiary thereof.

Any of the above markers may therefore be correlated, by carrying outthe cytocapacity test as described in the above example 1, and using asan additional parameter the marker as described above. The additionalparameter is then correlated with the hematopoietic recovery in a likemanner as the leukocyte count, the correlation of which to hematopoieticrecovery is described in example 1 hereinabove. A correlation betterthan that of leukocyte count with hematopoietic recovery, and/or withthe therapy- and outcome-related factors as described in the examplesherein, is preferred.

EXAMPLE 8 The Cytocapacity Test in a Group of Patients With MultipleMyeloma or Relapsed Lymphoma

Patients and Methods

Patients

The investigation was performed in 86 patients with multiple myeloma(MM) or relapsed lymphoma (LYM). 49 patients (57%) were male, 37patients (43%) were female. The median age was 53 years (range 18-68years). Before high-dose therapy, the patients had received a median of6 cycles of chemotherapy (range 0-25 cycles). Radiation therapy had beengiven to 29 patients (34%). The patients gave informed consent to theirtreatment. This patient cohort partially overlaps with the patientcohort of Examples 1-6.

Blood Stem Cell Mobilization, Harvesting, Processing andCryopreservation

In the vast majority of patients, the IEV regimen with G-CSF was usedfor stem cell mobilization. The IEV regimen consists of ifosfamide 2500mg/m² intravenously day 1-3, epirubicin 100 mg/m² intravenously day 1and etoposide 150 mg/m² intravenously day 1-3 followed by G-CSF(filgrastim; Amgen, Thousand Oaks, Calif., USA) at a dose of 5 μg/kgsubcutaneously daily from day 5 until the completion of blood stem cellharvesting. Apart from individual dose reductions, patients with MM andage >=60 years received IEV in a 75% dosage since 2000. PBSCs wereharvested when the post-nadir, G-CSF stimulated leukocyte count rose upto 5000-10000/μL or above using a COBE Spectra (COBE, Heimstetten,Germany) or an AS104 (Fresenius, St. Wendel, Germany) cell separator andstandard programs. In some cases, harvested blood stem cells from asingle leukapheresis underwent immunomagnetic B cell purging (MaxSep,Baxter Immunotherapy, Unterschleissheim, Germany) immediately after thecollection. The blood stem cells were mixed with an equal volume of afreezing solution which was prepared with 5% HSA and 100% DMSO(Cryoserv, Tera Pharmaceuticals, Midvale, Utah, USA) (4:1). The finalDMSO concentration was 10%. After computerized controlled-rate freezing,the bags containing the blood stem cells were stored in the vapor phaseof liquid nitrogen.

CD34⁺ Cell Enumeration in the Blood Stem Cell Autograft

The determination of CD34⁺ cells was carried out according to theguidelines of ISHAGE (Sutherland et al. 1996) using a FACScan (BD,Mountain View, Calif., USA) or an EPICS XL-MCL (Electronics, Miami,Fla., USA) flow cytometer equipped with an argon laser. Whole blood wasincubated for 30 minutes at 4° C. in the dark with the PE conjugatedmonoclonal anti-CD34 antibody and the FITC conjugated monoclonalanti-CD45 antibody followed by a wash and red blood cell lysis (BD).Before 1998, 20000 cells were analysed, 75000 cells thereafter. Toexclude cell debris, platelets, remaining red cells and all CD45negative cells, a forward scatter versus CD45 fluorescence dot plot wasused. The double-positive CD34⁺/CD45⁺ cell population was then definedand backgated for low CD45 expression and low side scatter properties.The percentage of the so defined CD34⁺ cells was multiplied with thetotal nucleated cell content of the apheresis product to result in theabsolute number of CD34⁺ cells harvested. The nucleated cell content wasdetermined by automated cell counting using a Coulter STKS (Coulter,Miami, Fla., USA). The anti-CD34 antibody (HPCA-2), the anti-CD45antibody (2D1) and the isotype controls used were from BD.

High-dose Therapy and Blood Stem Cell Transplantation

One hundred and twenty-eight high-dose chemotherapy courses in the 86patients were investigated. Melphalan 200 mg/m² (MEL200) (Barlogie etal. 1997) was applied in 88 procedures (69%), melphalan 100 mg/m²(MEL100) (Palumbo et al. 1999) in 18 procedures (14%) and BUCY in 22procedures (17%) (Schiller et al. 1994; Weaver et al. 1999). Autologousblood stem cell transplantation was performed 48 hours after the lastdose of chemotherapy.

Application of G-CSF and Blood Cell Counts

The recombinant human G-CSF given subcutaneously after high-dosechemotherapy was filgrastim (Amgen, Thousand Oaks, Calif., USA) in 121cases and lenograstim (Chugai, Japan) in 7 cases. The single G-CSFinjection evaluated was given on the evening before autologous bloodstem cell transplantation at a dose of 5 μg/kg, approximately 30 hoursafter the last chemotherapy infusion. The induced WBC peaks weremeasured with the routine blood tests on the next morning, 12-14 hoursafter the single G-CSF dose. The autologous blood stem celltransplantation was carried out approximately 2 hours after the routineblood test. From the day after transplantation, G-CSF was given daily ata dose of 5 μg/kg until post-nadir WBC counts were between 5000/μL and10000/μL. Routine blood cell counts including a differential wereperformed on a Coulter STKS (Coulter, Miami, Fla., USA) daily from thestart of high-dose therapy until the patients left the hospital.

Supportive Care

All the patients were hospitalized during high-dose therapy, autograftand the post-transplant phase and received the same supportive care. Theantimicrobial prophylaxis consisted of the following: From the time ofautograft until neutrophil recovery ciprofloxacin 2×250 mg daily wasgiven. Oral amphotericin B suspension 4×1 mL was given during the entirehospital stay or alternatively, in case of oral amphotericinintolerance, oral fluconazole 1×100 mg daily. Pneumocystis cariniipneumonitis prophylaxis was given during the administration of high-dosechemotherapy with trimethoprim/sulfamethoxazole 160 mg/800 mg 2×1 daily,was stopped before transplantation and was continued after neutrophilrecovery with 2×1 on two consecutive days per week for 6 months. In caseof trimethoprim/sulfamethoxazole intolerance, alternatively apentamidine inhalation with 300 mg was performed every 4 weeks for 6months from autograft. Intravenous acyclovir was given at a dose of2×500 mg from the start of high-dose therapy until neutrophil recovery.Red blood cell products and single-donor platelets were substituted tomaintain a hemoglobin level above 80 g/L and a platelet-count above10000/μL. All blood cell products were CMV negative, were irradiatedwith 30 Gy and were transfused through a leukocyte reduction filter.Empirical intravenous antimicrobial therapy was started duringneutropenia after a single oral temperature >38.5° C. or whenfever >38.0° C. was present over at least one hour and was carried outaccording to previously published guidelines (Hughes et al. 1997). Theinitial treatment was carried out with piperacillin/tazobactam plusgentamicin. Escalation was performed with meropenem/vancomycin. If feverpersisted on days 5-7, intravenous amphotericin B was added. Theantimicrobial treatment was continued until neutrophil recovery and atleast 24 hours after the resolution of fever. Patients were dischargedfrom the hospital after neutrophil and platelet recovery and after thecessation of antimicrobial treatment.

Assessment of Hematopoietic Recovery and Infection

Neutrophil recovery was defined as the first day with a neutrophil countabove 500/μL from the day of autograft. Platelet recovery was defined asthe first day with an unsubstituted platelet count above 20000/μL fromthe day of autograft. The observation period was from high-dose therapyuntil the patients left the hospital. The assessment of infection wascarried out according to previously published criteria (Link et al.1994).

Objectives and Statistical Analysis

The objectives of this investigation were to correlate the induced WBCpeak as the indicator of the G-CSF reaction with the neutrophil andplatelet recovery and the rate and type of infection and to comparethese correlations with those obtained for the CD34⁺ cell number in theautologous blood stem cell autograft. All analyses presented (except forthe demographic and disease baseline characteristics) are based ontransplantation courses as units of observation.

The rates of neutrophil and platelet recovery over time were estimatedusing the product-limit method according to Kaplan and Meier (Kaplan andMeier, 1958) and prognostic subgroups were compared by use of thelog-rank test (Peto and Peto, 1972). Differences in continuous variablesbetween prognostic groups were tested by Wilcoxon-Mann-Whitney orKruskal-Wallis analysis, depending on the number of groups. In case ofnominal or ordered categories, Fisher's exact or chi² tests for trendwere applied. In order to examine the independent impact of thepredictive factors with regard to the different end-points, multivariateanalyses were carried out with stratified versions of the respectivetests, Cox (Cox, 1972) or logistic regression models depending on thenature of the outcome variable. All reported p-values result fromtwo-sided tests.

The Host G-CSF Reaction

A single subcutaneous G-CSF injection (5 μg/kg) was administered earlyafter 128 high-dose chemotherapy courses in 86 patients with multiplemyeloma or lymphoma. At this time point, the median WBC count was4100/μL (range 1800-10700/μL) and the median platelet count was197000/μL (range 24000-640000/μL). The G-CSF induced WBC peak wasmeasured 12-14 hours later. These transient WBC peaks had a differentmagnitude (FIG. 4) and consisted of around 90% neutrophils. The medianWBC peak was 17400/μL (range 3300-60600/μL). The distribution of WBCpeaks is shown in FIG. 5A. Colony-forming cells or CD34⁺ cells were notdetected within these WBC peaks. After the WBC peak, the WBC countsdeclined and severe leukopenia (<200/μL) followed in all cases.

Prediction of the Hematopoietic Recovery

Autologous blood stem cell transplantation was performed after themeasurement of the G-CSF reaction with a median of 3.92×10⁶ CD34⁺cells/kg (range 0.9-21.2). The time to neutrophil engraftment (>500/μL)was a median of 9 days (range 8-12) and the time to platelet engraftment(>20000/μL) was a median of 10 days (range 8-25).

The G-CSF reaction predicted the neutrophil (p<0.0001) and the plateletengraftment (p<0.0001) (table 4). The number of transplanted CD34⁺ cellsalso predicted the neutrophil (p<0.0001) and platelet engraftment(p<0.0001), as expected. Other patient or treatment characteristics andthe WBC count before G-CSF did not correlate with the hematopoieticrecovery (table 4). The correlation between the G-CSF reaction and theCD34⁺ cell number was weak (r=0.21; p=0.02). In multivariate analysis ina Cox model, the prediction of the hematopoietic recovery by the G-CSFreaction was independent from the effect of transplanted CD34⁺ cells(table 5).

Prediction of Infection

Fever >38.0° C. was present in 69 of 128 procedures (54%). Infectionswere documented in 29 procedures (23%). The G-CSF reaction highlysignificantly predicted the rate of documented infections (p<0.0001).Leukopenia <1000/μL lasted for a median of 5 days (range 3-11). As couldbe expected, also the duration of leukopenia correlated with the rate ofdocumented infections (p=0.003). In multivariate analysis performed as alogistic regression, the G-CSF reaction was independent from leukopeniaduration in the prediction of infection and emerged as the dominantprognostic factor (p<0.0001) (table 5). There was no correlation betweenthe rate of documented infection and the number of transplanted CD34⁺cells or other patient and treatment characteristics (table 4). A numberof factors correlated with the occurrence of fever, but none of theseretained independent significance in multivariate analysis.

The vast majority of documented infections (86%) occurred at a low G-CSFreaction (FIG. 5 and table 6). Bacterial isolates from the blood streamwere predominantely coagulase-negative staphylococci. Also, the majorityof pneumonias (83%) and all the invasive fungal infections andtreatment-related deaths were associated with a low G-CSF reaction.

In 39 patients where a first and second high-dose chemotherapy coursewas analysed, the G-CSF reaction decreased from a median of 21700/μLWBC's (range 6400-59300/μL) after the first to a median of 15600/μLWBC's (range 3300-24800/μL) after the second high-dose chemotherapy. Atthe same time, the proportion of documented infection increased by 38%.TABLE 4 Univariate analysis of the association of factors with stem cellengraftment and risk of infection. Engraftment Infection ANC >500/μLPLT >22.000/μL Documented Age 0.19 0.40 0.53 (≦53 vs. >53 years) Gender0.96 0.53 0.68 (male vs. female) Diagnosis 0.96 0.83 0.76 (MM vs. LYM)Previous Chemotherapy 0.14 0.81 0.40 (≦6 vs. >6 cycles) PreviousRadiation 0.52 1.00 0.65 (Yes vs. No) CD34 + cells transplanted <0.0001<0.0001 0.79 (<2.5 vs. 2.5-5.0 vs. >5.0) G-CSF reaction <0.0001 <0.0001<0.0001 (1. vs. 2. vs. 3./4. quartile) WBC count before G-CSF 0.06 0.540.20 (<4000/μL vs. ≧4000/μL) Days with leukocytes <1000/μL — — 0.003 (≦5days vs. >5 days) p-valueANC = Absolute Neutrophil Count;PLT = Platelet Count;MM = multiple myeloma;LYM = lymphoma.

TABLE 5 Multivariate analysis of relevant factors for stem cellengraftment and documented infections. Engraftment ANC >500/μlPLT >20.000/μl CD34 +cells transplanted <0.0001 <0.0001 G-CSF reaction0.02 0.0006 p-value Documented infections G-CSF reaction <0.0001 Dayswith leukocytes <1.000/μL >0.2 p-value

TABLE 6 Correlation between G-CSF reaction and type of documentedinfection. Quartile 1 Quartile 2 Quartile 3 Quartile 4 G-CSF reaction N32% 32% 32% 32% Blood stream infection 22 12 3 0 Coag.-neg. Staphyl. 199 3 Streptococcus 3 Propionibacterium 3 Pneumonia 25 6 6 0 Invasivefungal infection 6 0 0 0 Severe enterocolitis 3 6 0 0 Other infections12 6 6 0 Sinusitis 3 3 Cholecystitis 3 Soft-tissue infection 6 Myelitis3 Catheter entry-site 3 infection Port-sepsis 3 Overall incidence 47 3112 0 Treatment-related 6 0 0 0 mortality

In this investigation we found that the reaction of the host to a singledose of pharmacological G-CSF before a phase of myelosuppression followscan be a predictor of critical hematopoietic functions during the courseand for overcoming myelosuppression. This highlights the potential ofG-CSF and suggests that G-CSF could be used in a diagnostic procedure.To assess and predict the capacity of host defense mechanisms to fightinfection and to compensate cytopenia before a manifestation ofmyelosuppression has not been possible so far. The G-CSF reactionprovokes a leukocyte mobilization by targeting the bone marrowmicroenvironment and directly represents an effector cell response. Thisis conceptually different from measuring cytokine levels. Ourinvestigation suggests a microenvironmental capacity of host defensewhich apparently can be targeted and tested by pharmacological G-CSF invivo.

The number of transplanted CD34⁺ cells is the major relevant factor forstem cell engraftment after high-dose chemotherapy (Bensinger et al.1995; Tricot et al. 1995; Weaver et al. 1995; Ketterer et al. 1988).This was confirmed in our study. The G-CSF reaction, however, predictedstem cell engraftment independently from the number of transplantedCD34⁺ cells. The G-CSF reaction therefore represents an importantindependent factor for the hematopoietic recovery which is maintainedduring reformation of a functional hematopoietic microenvironmentfollowing autologous stem cell transplantation.

The duration of leukopenia is a major known factor for the risk ofinfection in the myelosuppressed host (Bodey et al. 1966). This could berecapitulated in our study. The G-CSF reaction, however, was independentfrom leukopenia duration in the prediction of infection and emerged asthe dominant prognostic factor (p<0.0001). G-CSF is the central mediatorin the host response to neutropenia and infection (Watari et al. 1989;Kawakami et al. 1990). The host leukocyte response provoked bypharmacological G-CSF possibly was a premature reflection of thepotential of this G-CSF-inducible “emergency reaction” duringmyelosuppression. For the transplanted CD34⁺ cell number, no correlationwith the rate of documented infections was found (p=0.79). As long as aregular neutrophil engraftment occurs, there seems to be no furtherimpact of CD34⁺ cell number on the rate of infection. Experiments inmice reveal the influence of G-CSF on microenvironmental functionality.In transgenic mice constitutively expressing high levels of human G-CSF,megakaryocytopoiesis in the bone marrow was augmented although G-CSF hasnot been assigned a specific role in megakaryocytopoiesis (Fujita et al.2001). In transplantation experiments, it was shown that thisaugmentation of megakaryocytopoiesis was dependent on microenvironmentalchanges induced by transgenic human G-CSF. This qualitatively alteredmicroenvironment allowed a faster platelet recovery followinghematopoietic transplantation. This fits to our clinical finding thatthe G-CSF reaction is an independent predictor of platelet recovery. InG-CSF knock-out mice, which did not express G-CSF, a chronicneutropenia, a diminution of progenitor cells, a reduced fastmobilizability of leukocytes by a single pharmacological dose of G-CSFand a higher susceptibility to experimental infection was found(Lieschke et al. 1994). These findings in part correspond to ourobservations in patients with a low G-CSF reaction. One could speculatethat developmental effects of G-CSF on the microenvironment arereflected in the G-CSF reaction.

The high-dose chemotherapy setting likely favored the identification ofthe predictive potential of the G-CSF reaction because of the highdegree of myelosuppression, the associated higher number of infectiousepisodes and the close monitoring of patients in the hospital. Incontrast to the passive observation of a neutrophil nadir duringmyelosuppression after chemotherapy to draw conclusions aboutneutropenic events in the further course of therapy (Silber et al.1998), the G-CSF reaction can be assessed at normal WBC counts andobviously is dependent on the presence of mobilizable leukocytes in thebone marrow. The in part very high leukocyte peaks and their highvariability in our investigation suggest a maximum mobilizing effectwhich allows to recognize differences between patients. The high-dosechemotherapy given immediately before probably exerted a priming effectfor the mobilization with G-CSF, since the observed strong mobilizingeffects usually are not observed at a G-CSF dose of 5 μg/kg. The effectsof pharmacological G-CSF are dose-dependent. A strong mobilizing effectduring steady-state hematopoiesis or after conventional chemotherapy isobserved only at G-CSF doses of 10 μg/kg or above (Morstyn et al. 1988;Morstyn et al. 1989).

About one third of autologous blood stem cell transplants worldwide areperformed in multiple myeloma. The vast majority of these patientsreceives melphalan or busulfan/cyclophosphamide high-dose chemotherapybefore transplantation, as it was applied in our investigation. In thesepatients, the G-CSF reaction could provide a basis to administerrisk-stratified supportive care in controlled clinical trials(Meisenberg et al. 1997; Herrmann et al. 1999; Kern et al. 1999;Freifeld et al. 1999). Moreover, the G-CSF reaction could play a role inother areas like chemotherapy without stem cell transplantation forsolid tumors. It would be interesting to investigate whether the actualneed for pharmacological G-CSF could be determined on the basis of theG-CSF reaction. The G-CSF reaction also could contribute to thedevelopment of novel prophylactic strategies against infection(Noursadeghi et al. 2002).

EXAMPLE 9 The Cytocapacity Test Predicts the Duration of Leukopenia andNeutropenia

The following analysis was done on the cases of example 8. Thecytocapacity test (WBC peak) predicts the duration of leukopenia andneutropenia (table 7). TABLE 7 Correlation of the duration of leukopeniaand neutropenia with the cytocapacity test (WBC peak) and the number oftransplanted CD34⁺ cells. WBC peak CD34⁺ cells × 10⁶/kg Quartile 1 2 3 4<2.5 2.5-5.0 >5.0 Leukopenia < p < 0.001 p < 0.001 1.000/μL (Days)Minimum 4 4 3 3 5 4 3 25. Percentile 6 5 5 5 6 5 4 50. Percentile 6 6 55 6 6 5 75. Percentile 7 6 6 5 7 6 5 Maximum 11 9 7 8 11 8 8 Neutropenia< 500/μL p < 0.001 p < 0.001 (Days) Minimum 4 4 3 3 4 3 3 25. Percentile5 5 4 4 6 5 4 50. Percentile 6 5 5 5 6 5 5 75. Percentile 7 6 6 5 7 6 5Maximum 9 9 7 7 9 8 7

EXAMPLE 10 The Leukocyte or White Blood Cell (WBC) Peak as the Result ofthe Cytocapacity Test is Very Similar to the Respective Neutrophil Peak

The following data (table 8) is based on the cases of example 8. TABLE 8Correlation between the induced WBC and the neutrophil peak. WBC Peak(/μL) Neutrophil Peak (/μL) Minimum 3.300 3.100 25. Percentile 12.80012.100 50. Percentile 17.400 16.700 75. Percentile 23.300 22.400 Maximum60.600 56.300 r = 0.998

EXAMPLE 11 The Cytocapacity Test After Myelosuppressive ChemotherapyWithout Hematopoietic Stem Cell Transplantation

Forty-eight patients with lymphoma or multiple myeloma received the IEVchemotherapy followed by G-CSF for tumor reduction and stem cellmobilization as specified in Example 8. The first G-CSF injection wasgiven on day 5 of the regime and the induced leukocyte peak on day 6 wastaken as the cytocapacity test. The leukocyte count rose from a medianof 5.000/μL (range 1.500-8.200/μL) to a median of 10.100/μl (range300-46.700/μL). The graduation of the leukocyte peak predicted the riskof fever and infection (p<0.05) (table 9) during the following phase ofmyelosuppression. A hematopoietic transplantation was not carried out.TABLE 9 Correlation of the cytocapacity test with fever and infectionafter myelosuppressive chemotherapy with the IEV regime. Leukocyte peak(=cytocapacity test) N Fever and infection <=median 23 7 (30%)p<0.05 >median 23 1  (4%)References

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1. A method for determining the hematopoietic cytocapacity of a subjectcomprising the steps of: a) determining the amount of leukocytes presentin a blood sample obtained from a subject, wherein said subject has beensubjected to administration of a single dose of G-CSF sufficient toallow mobilization or release of the leukocytes from hematopoieticproduction and storage tissues and sites of margination into the blood;and b) determining the hematopoietic cytocapacity by assessing theamount of leukocytes determined in step (a) with the amount ofleukocytes which have been mobilized or released in a control subjectwherein said control subject is selected from the group consisting ofsubjects having (i) a high risk for a disease, disorder or complicationassociated with high-dose cytotoxic chemotherapy and/or hematopoieticcell transplantation, (ii) an intermediate risk for a disease, disorderor complication associated with high-dose cytotoxic chemotherapy and/orhematopoietic cell transplantation or (iii) a low risk for a disease,disorder or complication associated with high-dose cytotoxicchemotherapy and/or hematopoietic cell transplantation.
 2. A method forselecting a suitable antimicrobial prophylaxis or therapy for a subject,wherein said method comprises the steps of the method of claim 1 and thefurther step (c) selecting a suitable antimicrobial prophylaxis ortherapy for said subject based on the results obtained in step (b).
 3. Amethod according to claim 2, wherein said prophylaxis or therapy is aprophylaxis or therapy for the treatment, prevention or amelioration ofan infection.
 4. A method according to claim 3, wherein said infectionis selected from the group of fungal, viral, protozoal, parasitical andbacterial infections.
 5. A method according to claim 3, wherein saidinfection is selected from the group consisting of pneumonia, invasivefungal infection, enterocolitis, soft-tissue infection, and sepsis.
 6. Amethod for selecting a suitable prophylaxis or therapy for neutropenicfever for a subject, wherein said method comprises the steps of themethod of claim 1 and the further step (c) selecting a suitableprophylaxis or therapy for neutropenic fever for said subject based onthe results obtained in step (b).
 7. A method for selecting a suitableamount of hematopoietic stem cells to be transfused for the therapy of asubject, wherein said method comprises the steps of the method of claim1 and the further step (c) of selecting the amount of said cells to betransfused for the therapy of a subject based on the results obtained instep (b).
 8. A method for selecting a suitable amount of a hematopoieticgrowth factor or cytokine for the treatment of a subject, wherein saidmethod comprises the steps of the method of claim 1 and the further step(c) selecting a suitable amount of a hematopoietic growth factor orcytokine for the treatment of said subject based on the results obtainedin step (b).
 9. A method of preparing a diagnostic composition fordiagnosing a susceptibility for a disease, disorder or complicationassociated with high-dose cytotoxic chemotherapy and/or hematopoieticcell transplantation in a subject comprising utilizing leukocytesobtained from said subject, wherein said subject has been subjected toadministration of a dose of G-CSF sufficient to allow mobilization orrelease of the leukocytes from hematopoietic production and storagetissues and sites of margination into the blood.
 10. The method of claim1 or claim 9, wherein said disease, disorder or complication associatedwith high-dose cytotoxic chemotherapy, hematopoietic celltransplantation, or both is neutropenic fever, microbial infection,delayed hematopoietic recovery, bleeding, immunosuppression,immunological effects directed against the host, high level ofsupportive care, morbidity and mortality.
 11. The method of claim 1 orclaim 9, wherein said subject is a human.
 12. The method of claim 1 orclaim 9, wherein said subject has been subjected to high-dosechemotherapy.
 13. The method of claim 12, wherein said high-dosechemotherapy comprises administration of melphalan, busulfan,cyclophosphamide, carmustine, etoposide, or cytarabine.
 14. The methodof claim 1 or claim 9, wherein said subject has been subjected tomyelosupressive chemotherapy.
 15. The method of claim 14, wherein saidmyelosupressive therapy comprises the administration ofcyclophosphamide, etoposide, carmustine, cytarabine, melphalan,busulfan, doxorubicin, epirubicin, paclitaxel, docetaxel, thiotepa,fludarabine, vincristine, bendamustine, cisplatin, carboplatin,daunorubicin, fluorouracil, gemcitabine, idarubicin, ifosfamide,irinotecan, methotrexate, mitoxantrone, oxaliplatin, treosulfan,vinblastine, or vinorelbine.
 16. The method of claim 1 or claim 9,wherein said subject has been subjected to radiotherapy, or suffers froma primary or secondary bone marrow disease, an autoimmune disease, ahereditary disease or disorder or an infection.
 17. The method of claim1 or claim 9, wherein said G-CSF is filgrastim or lenograstim.
 18. Themethod of claim 1 or claim 9, wherein said dose of G-CSF is selectedfrom a range of 1 to 20 μg/kg body weight of the G subject.
 19. Themethod of claim 1 or claim 9, wherein said dose of G-CSF is 1.0, 2.5, 5,7.5, or 10 μg/kg body weight of the subject.
 20. The method of claim 1or claim 9, wherein said time sufficient to allow mobilization orrelease of the leukocytes is in the range of 1 to 120 hours.
 21. Themethod of claim 1 or claim 9, wherein said time sufficient to allowmobilization or release of the leukocytes is at least 1 hour, at least 2hours, at least 6 hours, at least 10 hours, at least 12 hours, at least14 hours or at least 18 hours.